The intestinal commensal fungus Wallemia mellicola enhances asthma in mice through Dectin-2.

Overgrowth of the fungus Wallemia mellicola in the intestines of mice enhances the severity of asthma. Wallemia mellicola interacts with the immune system through Dectin-2 expressed on the surface of myeloid and intestinal epithelial cells. Using Dectin-2-deficient mice, we show that the interaction of W. mellicola with Dectin-2 is essential for the gut-lung pathways, enhancing the severity of asthma in mice with W. mellicola intestinal dysbiosis. These findings offer better insight into dysbiosis-associated inflammation and highlight the role pattern recognition receptors have in immune recognition of commensal fungi in the gut, leading to alterations in immune function in the lungs.

Targeting Pulmonary Fibrosis by SLC1A5-Dependent Glutamine Transport Blockade.

The neutral amino acid glutamine plays a central role in TGF-ß (transforming growth factor-ß)-induced myofibroblast activation and differentiation. Cells take up glutamine mainly through a transporter expressed on the cell surface known as solute carrier SLC1A5 (solute carrier transporter 1A5). In the present work, we demonstrated that profibrotic actions of TGF-ß are mediated, at least in part, through a metabolic maladaptation of SLC1A5 and that targeting SLC1A5 abrogates multiple facets of fibroblast activation. This approach could thus represent a novel therapeutic strategy to treat patients with fibroproliferative diseases. We found that SLC1A5 was highly expressed in fibrotic lung fibroblasts and fibroblasts isolated from idiopathic pulmonary fibrosis lungs. The expression of profibrotic targets, cell migration, and anchorage-independent growth by TGF-ß required the activity of SLC1A5. Loss or inhibition of SLC1A5 function enhanced fibroblast susceptibility to autophagy; suppressed mTOR, HIF (hypoxia-inducible factor), and Myc signaling; and impaired mitochondrial function, ATP production, and glycolysis. Pharmacological inhibition of SLC1A5 by the small-molecule inhibitor V-9302 shifted fibroblast transcriptional profiles from profibrotic to fibrosis resolving and attenuated fibrosis in a bleomycin-treated mouse model of lung fibrosis. This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of glutamine transport in fibrosis, providing a framework for new paradigm-shifting therapies targeting cellular metabolism for this devastating disease.

Dysbiosis of the intestinal fungal microbiota increases lung resident group 2 innate lymphoid cells and is associated with enhanced asthma severity in mice and humans.

BACKGROUND: The gut-lung axis is the concept that alterations of gut microbiota communities can influence immune function in the lungs. While studies have explored the relationship between intestinal bacterial dysbiosis and asthma development, less is understood about the impact of commensal intestinal fungi on asthma severity and control and underlying mechanisms by which this occurs. METHODS: Wild-type mice were treated with Cefoperazone to deplete gut bacteria and administered Candida albicans or water through gavage. Mice were then sensitized to house dust mite (HDM) and their lungs were analyzed for changes in immune response. Humans with asthma were recruited and stool samples were analyzed for Candida abundance and associations with asthma severity and control. RESULTS: Mice with intestinal Candida dysbiosis had enhanced Th2 response after airway sensitization with HDM, manifesting with greater total white cell and eosinophil counts in the airway, and total IgE concentrations in the serum. Group 2 innate lymphoid cells (ILC2) were more abundant in the lungs of mice with Candida gut dysbiosis, even when not sensitized to HDM, suggesting that ILC2 may be important mediators of the enhanced Th2 response. These effects occurred with no detectable increased Candida in the lung by culture or rtPCR suggesting gut-lung axis interactions were responsible. In humans with asthma, enhanced intestinal Candida burden was associated with the risk of severe asthma exacerbation in the past year, independent of systemic antibiotic and glucocorticoid use. CONCLUSIONS: Candida gut dysbiosis may worsen asthma control and enhance allergic airway inflammation, potentially mediated by ILC2. Further studies are necessary to examine whether microbial dysbiosis can drive difficult-to-control asthma in humans and to better understand the underlying mechanisms.

Mechanism of regulation of 'chromosome kissing' induced by Fob1 and its physiological significance.

Protein-mediated "chromosome kissing" between two DNA sites in trans (or in cis) is known to facilitate three-dimensional control of gene expression and DNA replication. However, the mechanisms of regulation of the long-range interactions are unknown. Here, we show that the replication terminator protein Fob1 of Saccharomyces cerevisiae promoted chromosome kissing that initiated rDNA recombination and controlled the replicative life span (RLS). Oligomerization of Fob1 caused synaptic (kissing) interactions between pairs of terminator (Ter) sites that initiated recombination in rDNA. Fob1 oligomerization and Ter-Ter kissing were regulated by intramolecular inhibitory interactions between the C-terminal domain (C-Fob1) and the N-terminal domain (N-Fob1). Phosphomimetic substitutions of specific residues of C-Fob1 counteracted the inhibitory interaction. A mutation in either N-Fob1 that blocked Fob1 oligomerization or C-Fob1 that blocked its phosphorylation antagonized chromosome kissing and recombination and enhanced the RLS. The results provide novel insights into a mechanism of regulation of Fob1-mediated chromosome kissing.

Transforming growth factor beta induces fibroblasts to express and release the immunomodulatory protein PD-L1 into extracellular vesicles.

Transforming growth factor-beta (TGFß) is an enigmatic protein with various roles in healthy tissue homeostasis/development as well as the development or progression of cancer, wound healing, fibrotic disorders, and immune modulation, to name a few. As TGFß is causal to various fibroproliferative disorders featuring localized or systemic tissue/organ fibrosis as well as the activated stroma observed in various malignancies, characterizing the pathways and players mediating its action is fundamental. In the current study, we found that TGFß induces the expression of the immunoinhibitory molecule Programed death-ligand 1 (PD-L1) in human and murine fibroblasts in a Smad2/3- and YAP/TAZ-dependent manner. Furthermore, PD-L1 knockdown decreased the TGFß-dependent induction of extracellular matrix proteins, including collagen Iα1 (colIα1) and alpha-smooth muscle actin (α-SMA), and cell migration/wound healing. In addition to an endogenous role for PD-L1 in profibrotic TGFß signaling, TGFß stimulated-human lung fibroblast-derived PD-L1 into extracellular vesicles (EVs) capable of inhibiting T cell proliferation in response to T cell receptor stimulation and mediating fibroblast cell migration. These findings provide new insights and potential targets for a variety of fibrotic and malignant diseases.

SIRT7-mediated modulation of glutaminase 1 regulates TGF-ß-induced pulmonary fibrosis.

In the current work we show that the profibrotic actions of TGF-ß are mediated, at least in part, through a metabolic maladaptation in glutamine metabolism and how the inhibition of glutaminase 1 (GLS1) reverses pulmonary fibrosis. GLS1 was found to be highly expressed in fibrotic vs normal lung fibroblasts and the expression of profibrotic targets, cell migration, and soft agar colony formation stimulated by TGF-ß required GLS1 activity. Moreover, knockdown of SMAD2 or SMAD3 as well as inhibition of PI3K, mTORC2, and PDGFR abrogated the induction of GLS1 by TGF-ß. We further demonstrated that the NAD-dependent protein deacetylase, SIRT7, and the FOXO4 transcription factor acted as endogenous brakes for GLS1 expression, which are inhibited by TGF-ß. Lastly, administration of the GLS1 inhibitor CB-839 attenuated bleomycin-induced pulmonary fibrosis. Our study points to an exciting and unexplored connection between epigenetic and transcriptional processes that regulate glutamine metabolism and fibrotic development in a TGF-ß-dependent manner.

IPF pathogenesis is dependent upon TGFß induction of IGF-1.

Pathogenic fibrotic diseases, including idiopathic pulmonary fibrosis (IPF), have some of the worst prognoses and affect millions of people worldwide. With unclear etiology and minimally effective therapies, two-thirds of IPF patients die within 2-5 years from this progressive interstitial lung disease. Transforming Growth Factor Beta (TGFß) and insulin-like growth factor-1 (IGF-1) are known to promote fibrosis; however, myofibroblast specific upregulation of IGF-1 in the initiation and progression of TGFß-induced fibrogenesis and IPF have remained unexplored. To address this, the current study (1) documents the upregulation of IGF-1 via TGFß in myofibroblasts and fibrotic lung tissue, as well as its correlation with decreased pulmonary function in advanced IPF; (2) identifies IGF-1's C1 promoter as mediating the increase in IGF-1 transcription by TGFß in pulmonary fibroblasts; (3) determines that SMAD2 and mTOR signaling are required for TGFß-dependent Igf-1 expression in myofibroblasts; (4) demonstrates IGF-1R activation is essential to support TGFß-driven profibrotic myofibroblast functions and excessive wound healing; and (5) establishes the effectiveness of slowing the progression of murine lung fibrosis with the IGF-1R inhibitor OSI-906. These findings expand our knowledge of IGF-1's role as a novel fibrotic-switch, bringing us one step closer to understanding the complex biological mechanisms responsible for fibrotic diseases and developing effective therapies.

Functional architecture of the Reb1-Ter complex of Schizosaccharomyces pombe.

Reb1 ofSchizosaccharomyces pomberepresents a family of multifunctional proteins that bind to specific terminator sites (Ter) and cause polar termination of transcription catalyzed by RNA polymerase I (pol I) and arrest of replication forks approaching the Ter sites from the opposite direction. However, it remains to be investigated whether the same mechanism causes arrest of both DNA transactions. Here, we present the structure of Reb1 as a complex with a Ter site at a resolution of 2.7 Å. Structure-guided molecular genetic analyses revealed that it has distinct and well-defined DNA binding and transcription termination (TTD) domains. The region of the protein involved in replication termination is distinct from the TTD. Mechanistically, the data support the conclusion that transcription termination is not caused by just high affinity Reb1-Ter protein-DNA interactions. Rather, protein-protein interactions between the TTD with the Rpa12 subunit of RNA pol I seem to be an integral part of the mechanism. This conclusion is further supported by the observation that double mutations in TTD that abolished its interaction with Rpa12 also greatly reduced transcription termination thereby revealing a conduit for functional communications between RNA pol I and the terminator protein.

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest.

Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1-Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2-7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2-7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2-7 had to be phosphorylated for binding to phospho-Tof1-Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2-7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1-Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1-Ter complex.

Vardenafil Activity in Lung Fibrosis and In Vitro Synergy with Nintedanib.

Idiopathic pulmonary fibrosis (IPF) remains an intractably fatal disorder, despite the recent advent of anti-fibrotic medication. Successful treatment of IPF, like many chronic diseases, may benefit from the concurrent use of multiple agents that exhibit synergistic benefit. In this light, phosphodiesterase type 5 inhibitors (PDE5-Is), have been studied in IPF primarily for their established pulmonary vascular effects. However, recent data suggest certain PDE5-Is, particularly vardenafil, may also reduce transforming growth factor beta 1 (TGF-ß1) activation and extracellular matrix (ECM) accumulation, making them a potential target for therapy for IPF. We evaluated fibroblast TGF-ß1-driven extracellular matrix (ECM) generation and signaling as well as epithelial mesenchymal transformation (EMT) with pretreatment using the PDE5-I vardenafil. In addition, combinations of vardenafil and nintedanib were evaluated for synergistic suppression of EMC using a fibronectin enzyme-linked immunosorbent assay (ELISA). Finally, the effects of vardenafil on fibrosis were investigated in a bleomycin mouse model. Our findings demonstrate that vardenafil suppresses ECM generation alone and also exhibits significant synergistic suppression of ECM in combination with nintedanib in vitro. Interestingly, vardenafil was shown to improve fibrosis markers and increase survival in bleomycin-treated mice. Vardenafil may represent a potential treatment for IPF alone or in combination with nintedanib. However, additional studies will be required.

Homologue of mammalian apolipoprotein A-II in non-mammalian vertebrates.

Although apolipoprotein with molecular weight 14 kDa (apo-14 kDa) is associated with fish plasma high-density lipoproteins (HDLs), it remains to be determined whether apo-14 kDa is the homologue of mammalian apoA-II. We have obtained the full cDNA sequences that encode Japanese eel and rainbow trout apo-14 kDa. Homologues of Japanese eel apo-14 kDa sequence could be found in 14 fish species deposited in the DDBJ/EMBL/GenBank or TGI database. Fish apo-14 kDa lacks propeptide and contains more internal repeats than mammalian apoA-II. Nevertheless, phylogenetic analysis allowed fish apo-14 kDa to be the homologue of mammalian apoA-II. In addition, in silico cloning of the TGI, Ensembl, or NCBI database revealed apoA-IIs in dog, chicken, green anole lizard, and African clawed frog whose sequences had not so far been available, suggesting both apoA-I and apoA-II as fundamental constituents of vertebrate HDLs.

Hexokinase 2 couples glycolysis with the profibrotic actions of TGF-ß.

Metabolic dysregulation in fibroblasts is implicated in the profibrotic actions of transforming growth factor-ß (TGF-ß). Here, we present evidence that hexokinase 2 (HK2) is important for mediating the fibroproliferative activity of TGF-ß both in vitro and in vivo. Both Smad-dependent and Smad-independent TGF-ß signaling induced HK2 accumulation in murine and human lung fibroblasts through induction of the transcription factor c-Myc. Knockdown of HK2 or pharmacological inhibition of HK2 activity with Lonidamine decreased TGF-ß-stimulated fibrogenic processes, including profibrotic gene expression, cell migration, colony formation, and activation of the transcription factors YAP and TAZ, with no apparent effect on cellular viability. Fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibited an increased abundance of HK2. In a mouse model of bleomycin-induced lung fibrosis, Lonidamine reduced the expression of genes encoding profibrotic markers (collagenΙα1, EDA-fibronectin, α smooth muscle actin, and connective tissue growth factor) and stabilized or improved lung function as assessed by measurement of peripheral blood oxygenation. These findings provide evidence of how metabolic dysregulation through HK2 can be integrated within the context of profibrotic TGF-ß signaling.

Mechanism of Regulation of Intrachromatid Recombination and Long-Range Chromosome Interactions in Saccharomyces cerevisiae.

The NAD-dependent histone deacetylase Sir2 controls ribosomal DNA (rDNA) silencing by inhibiting recombination and RNA polymerase II-catalyzed transcription in the rDNA of Saccharomyces cerevisiae Sir2 is recruited to nontranscribed spacer 1 (NTS1) of the rDNA array by interaction between the RENT ( RE: gulation of N: ucleolar S: ilencing and T: elophase exit) complex and the replication terminator protein Fob1. The latter binds to its cognate sites, called replication termini (Ter) or replication fork barriers (RFB), that are located in each copy of NTS1. This work provides new mechanistic insights into the regulation of rDNA silencing and intrachromatid recombination by showing that Sir2 recruitment is stringently regulated by Fob1 phosphorylation at specific sites in its C-terminal domain (C-Fob1), which also regulates long-range Ter-Ter interactions. We show further that long-range Fob1-mediated Ter-Ter interactions in trans are downregulated by Sir2. These regulatory mechanisms control intrachromatid recombination and the replicative life span (RLS).